Abstract (inglese)

Hydrogenases, key enzymes in hydrogen metabolism in several microorganisms, are considered as a possible future energy source. In particular, the ability of the green alga C. reinhardtii to reduce protons and release hydrogen gas (H2) upon illumination by means of a [FeFe]-hydrogenase represents a phenomenon of great scientific interest, because it holds the promise of generating energy from nature’s most plentiful resources: solar energy and water. However, the catalytic activity is strongly and irreversibly inhibited by the molecular oxygen produced during photosynthesis; furthermore, the algal hydrogenase is expressed at very low levels and only in conditions of strict anaerobiosis. This mutually exclusive nature of O2 and H2 photoproduction cannot be easily overcome and represents an important problem in the development of H2 production biotechnology. The study of the structure-function relationship of [FeFe] hydrogenases, which would help to clarify the molecular mechanisms underlying both H2 production and O2 sensitivity, requires the characterization of purified native and modified proteins. The 3D structure of the [FeFe]-hydrogenase from C. reinhardtii has not been solved, mainly because of the very low levels of protein which can be directly purified from the algae. I overexpressed this enzyme in homologous and heterologous systems to obtain enough protein for biochemical studies. The cyanobacterium Synechococcus PCC 7942, which holds a bidirectional [NiFe]-hydrogenase, is able to produce the [FeFe]-hydrogenase from C. pasteurianum in a catalytically active form, thus suggesting that the [NiFe]-hydrogenase maturation pathway of cyanobacteria may be flexible enough to allow the biosynthesis of functional Fe-only enzyme. I obtained two constructs to stably transform the cyanobacterium Synechocystis sp. PCC 6803 and to enable it to express the C. reinhardtii [FeFe]-hydrogenase. The recombinant strains expressing the algal [FeFe]-hydrogenase were able to release H2 gas amounts 4 to 5 times higher than that of wild type strain (which has only a [NiFe]-hydrogenase). These data open new perspectives about the indispensable presence of HydE, HydF and HydG auxiliary proteins to obtain a correctly folded [FeFe]-hydrogenase. At the same time, I proceeded with the homologous overexpression of the [FeFe]-hydrogenase in C. reinhardtii. Since serious limits of this system are the low amount of protein expressed and the instability of mutant algal strain, I am defining new strategies to solve this problem. I will operate site-direct mutations in critical residues to understand the catalytic mechanism and to improve the hydrogen productivity of the enzyme.